The function of free sulfhydryl groups in the catalytic subunit of phosphorylase kinase

Date
1993
Authors
Yuan, Chiun-Jye
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Biochemistry, Biophysics and Molecular Biology
Abstract

Phosphorylase kinase, a serine/threonine type kinase, has been recently found to contain tyrosine kinase activity by phosphorylating angiotensin II. The tyrosine kinase activity of phosphorylase kinase requires Mn[superscript]2+. In the presence of saturable metal-nucleotide complex, MgATP, the serine kinase activity is inhibited by Mn[superscript]2+, while tyrosine kinase activity is activated. The tyrosine kinase activity in holoenzyme is Ca[superscript]2+-independent but can be regulated by autophosphorylation. One active site with two conformations is proposed; one favors seryl phosphorylation, and the second tyrosyl phosphorylation which is caused by the binding of second metal ion;The truncated [gamma] subunit of phosphorylase kinase is found to be inactivated by [mu]M range of Cu[superscript]2+. The inactivation can be readily reversed by DTT but not by EDTA or EGTA. This result suggests that inactivation is caused by disulfide bond formation. Two intramolecular disulfide bonds are formed by oxidation as determined by DTNB titration and non-reducing SDS-PAGE. The species containing the first disulfide bond is denoted as semi-oxidized form. Kinetic study of the semi-oxidized form, peptide mapping, and sequencing indicate that the first disulfide bond may be in or near the nucleotide binding domain of truncated [gamma]. The truncated [gamma] subunit can also be inactivated by a Cys-peptide dimer through thiol/disulfide exchange. The cross-linking of C-peptide to Cys-138 provides a steric hindrance and likely restricts the formation of an optimal conformation in the active site;To determine the function of each free sulfhydryl group all six thiols are replaced with serine by site-directed mutagenesis. In addition, C36A mutant was also prepared. All mutants except C36S and C184S show normal serine and tyrosine kinase activities. The kinetic studies show that Cys-36 influence ATP binding. It is suggested that mutation of Cys to Ser causes a conformational change and indirectly affects ATP binding. The mutant C184S is particularly interesting because its tyrosine but not serine kinase activity is affected by the mutation. Further studies show that C184S has a different substrate specificity from that of wild type truncated-[gamma] subunit. These results suggest that Cys-184 has an important role in the action of the truncated-[gamma] subunit on peptides containing tyrosyl residue.

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